Manfred D. Kessler

Spectrometric measurements in the anterior eye vasculature of the albino rabbit—A study with the EMPHO I

Intracapillary haemoglobin oxygenation (oxygen saturation) and haemoglobin concentration (corresponding with blood volume) were measured in different regions of the albino rabbit anterior eye segment using the Erlangen micro-lightguide spectrophotometer (EMPHO I). The tip of the microlightguide (active diameter 210 microns, cover diameter 0.5 mm) was placed in the vitreous by a goniometric mounting and controlled by a micromanipulator. After stepwise elevation of intraocular pressure (IOP) the haemoglobin oxygenation decreased slightly in the iris at an IOP-level of 60 mmHg, whereas in the three regions of the ciliary processes and in the peripheral choroid haemoglobin oxygenation did not decrease until values of 80 mmHg were applied. In contrast, haemoglobin concentration decreases when the IOP increases in all regions except in the pars plana where the haemoglobin concentration increased at 40 and 60 mmHg. The most pronounced reactive hyperaemia was found in the major ciliary processes. Quite passive changes of haemoglobin concentration were seen in the peripheral choroid. Effective regulatory mechanisms must exist which result in IOP-independent (20-60 mmHg) haemoglobin oxygenation, whereas pronounced changes in intracapillary haemoglobin concentration were observed. The decay of both parameters at IOP 80 mmHg is indicative of an exhaustion of their regulatory capacity. Epinephrine topically applied onto the conjunctival sac or injected into the common carotid artery led to a short-term decrease of haemoglobin concentration and, later, a hyperaemic response. A short-term haemoglobin deoxygenation was found especially in the major ciliary processes.

Spatial Distribution of Oxygen Supply Units in Heart and Skeletal Muscle and their Regulatory Significance

Investigations of local pO2 in skeletal and heart muscle revealed unexpected results. In both organs local pO2 measurements demonstrated that tissue oxygenation is structured in such a way that 10–15 characteristic O2 supply units do exist (see figures 1 and 2). Each individual supply unit shows a rather homogeneous distribution of the spatial pO2 profiles (Kessler et al. 1984). In skeletal muscle the size of the individual unit is approximately 400–600 μm in length with a diameter of approximately 150–200 μm. In such functional structures with relatively homogeneous patterns of capillary blood flow large pO2 gradients between the individual supply units are not induced. This prevents loss of oxygen by diffusion shunt.

Monitoring of Redox-State of Respiratory Enzymes and Myoglobin Oxygenation in the Working Rat Heart in Normoxia and Oxygen Deficiency

The cellular oxygen supply in the isolated, hemoglobin-free perfused, working rat heart can be determined by measurements of myoglobin oxygenation. However, for a precise analysis of mitochondrial hypoxia and anoxia (pO2 < 0.01 Torr) redox-state of respiratory enzymes must be known. By use of the EMPHO (Frank et al. 1989) it is possible to perform a high speed spectrometry within very small tissue volumes. Because of the characteristic absorption spectra of oxygenated and deoxygenated myoglobin and of the oxidized and reduced cytochrome aa3 within the wavelength interval from 500 to 630 nm it is possible to isolate these two pigments from the remission spectra and to determine the oxygenation state of myoglobin and the redox-state of cytochrom aa3.

Validation and improvements of an algorithm for the determination of hemoglobin oxygenations, based on spectral data recorded by a tissue spectrophotometer

The tissue spectrometer EMPHO allows measurements of absolute hemoglobinoxygenation values, noninvasive1y at any hemoglobin-perfused tissue, simply by applying visible light on the surface of theorgans under investigation. The hemoglobin oxygenation-algorithm is based on Kubelka-Munk-Theory fortackling both. absorbance and scattering phenomena. Broad-band tissue spectra ofbackscattered light serves as data basis for the analysis. In this study the algorithm was tested for measurements in highly scatteringmedia, in Intralipid©, where erythrocytes were added step by step. The hemoglobin concentration in the suspension varied from 0.01 to 1.0 [g hb Idl of suspension], which corresponds to the range of hemoglobin concentrations physiologically found in various types of tissue. The oxygenation was changed from 0 % to 100 % by using a hollow-fibre oxygenator. The costly study revealed that the algorithm works with high accuracy at a middlehemoglobin-concentration of 0.3 g/dl. The error of calculation was smaller than 2% of the absolute HbO value. The statistics proved that errors were larger at the highest and lowest values o hemoglobin concentration. It could clearly be shown that the errorcan be minimized to 1 % by application ofnew gold-standard hemoglobin spectra ofO % and 100 % oxygenation. Key words: tissue spectrometry, hemoglobin oxygenation, Kubelka-Munk Theory, light absorbance,light scattering, visible wavelength range.

Simultaneous monitoring of NAD(P)H, cytochromes, pO2, and HbO2 in liver tissue

A new spectrometer (EMPHO IIb) for measurements of NADH fluorescence spectra in tissue was developed which enables recording of 100 spectra per sec. by use of a rotating filter disc. The excitation of fluorescence is induced by a mercury lamp (HbO100 W/2) at 366nm. For the optical coupling of the instrument to the surface of tissues quartz microlight guides are applied.

Analysis of downregulation of cellular energy demand by 2D measurements of intracapillary HbO2, Hb, pO2, and redox state of cytochromes

Rapid microlightguide spectrometers (EMPHO IIa/b) and a multiwire pO2 electrode are applied for measurements of heterogeneous distribution of tissue oxygenation and redox state of respiratory enzymes in heart and rat liver. Optical and pO2 measurements are noninvasively performed by use of sensors placed on the surface of tissue. Measurements in isolated perfused rat and in dog heart in situ were performed in order to investigate the relation between myocardial oxygenation and function. The tissue monitoring in liver was initiated by optical and polarographic monitoring in the hemoglobin free perfused organ. Subsequently, erythrocytes were added to the perfusate in several steps. The experiments reveal clear evidence that a protective system of tissue is activated when critical pO2 values at the lethal corner of micro vessels fall off a critical threshold around 5 mmHg, thus causing a depletion of oxidative metabolism.

Noninvasive topographical investigation of functional parameters in the human skin

A rapid micro-lightguide spectrometer (EMPHO II) coupled to an automatic three axis positioning system enables very precise and fast 2D-scans at the surface of human skin. The positioning accuracy amounts to 1 micrometer. This allows measurements with excellent spatial reproducibility. With this system examinations of local distribution of HbO2 and Hb have been performed in human skin. For this purpose at the back of the hand areas of 5 by 5 mm to 5 by 10 mm were scanned in defined steps of 100 micrometers. Functional images of local hemoglobin concentration and hemoglobin oxygenation of microscopical structures have been resolved by use of 250 micrometer lightguide sensors. Two-dimensional-images of local oxygen supply parameters corresponding directly to morphological structures of human skin have been gained. The local pattern matches the distribution of the papillas of the corium. In the papillas the capillary loops supplying the lower part of the epidermis are situated. The measured parameters describe very exactly the local oxygen supply situation of the area under investigation.

Analysis of multiple-multipole scattering by time-resolved spectroscopy and spectrometry

Abstract not available.

Imaging of dynamic alteration of functional structures

Subcellular structures of liver are mainly caused by organelles of hepatocytes and endothelial cells when VIS and NIR are used for irradiation. Total tissue anoxia induces very fast shrinking of mitochondria by switching the oxygenator of the perfusion system from normoxia to total anoxia. This oxygen reaction causes a drastic decrease of backscattered light. For further investigations we used a protocol of three different conditions: 1. Stimulation of subcellular structures in Hb-free perfused liver by application of hypoxia and normoxia, 2. Effects of erythrocytes, 3. Measurements of tissue pigments. Investigations performed by use of our 2D- and 3D-imaging system revealed that the subcellular space with its organelles represents a highly dynamic system.

Oxygen supply (HbO2) in beating hearts of 14 coronary patients obtained by tissue monitoring with optical sensors

Local intra capillary HbO2 was monitored in beating hearts of 14 patients undergoing coronary bypass surgery. The spectra were measured in the epicardium of the left ventricle, supplied by the left coronary artery (LAD). All selected patients suffered form stenosis or occlusion of two to three vessels. The patients suffered from severe angina and showed hypokinesia in the angiography. Micro-light guide fibers with a diameter of 75micrometers were used for monitoring before and after bypass surgery. These light guides were connected to the Erlanger Micro Light guide Spectro Photometer EMPHO for registration. Local measurements were performed in the epicardium of the left ventricle in 25 areas 2.25cm2 each. Integrated gradient fields were plotted for each of the 14 patients before and after bypass surgery. The mean values of HbO2 in the respective areas were calculated and evaluated against the local value distribution.